Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction

Abstract

Multiparameter tissue imaging enables analysis of cell-cell interactions in situ, the cellular basis for tissue structure, and novel cell types that are spatially restricted, giving clues to biological mechanisms behind tissue homeostasis and disease. Here, we streamlined and simplified the multiplexed imaging method CO-Detection by indEXing (CODEX) by validating 58 unique oligonucleotide barcodes that can be conjugated to antibodies. We showed that barcoded antibodies retained their specificity for staining cognate targets in human tissue. Antibodies were visualized one at a time by adding a fluorescently labeled oligonucleotide complementary to oligonucleotide barcode, imaging, stripping, and repeating this cycle. With this we developed a panel of 46 antibodies that was used to stain five human lymphoid tissues: three tonsils, a spleen, and a LN. To analyze the data produced, an image processing and analysis pipeline was developed that enabled single-cell analysis on the data, including unsupervised clustering, that revealed 31 cell types across all tissues. We compared cell-type compositions within and directly surrounding follicles from the different lymphoid organs and evaluated cell-cell density correlations. This sequential oligonucleotide exchange technique enables a facile imaging of tissues that leverages pre-existing imaging infrastructure to decrease the barriers to broad use of multiplexed imaging.

Highly Multiplexed Phenotyping of Immunoregulatory Proteins in the Tumor Microenvironment by CODEX Tissue Imaging

“…All antibodies performed as expected when imaged during the CODEX multicycle experiment (Supplementary Figure 4), including the seven polyclonal antibodies used in this panel, which showed consistent staining intensity, appropriate spatial expression patterns, and no anomalous cross-reactivity. Importantly, the reproducibility of antibody staining is high within a single CODEX multicycle experiment for a given tissue region (16,18,21), between patient samples (Supplementary Figure 5) as well as across experiments processed in parallel (19). For all CODEX experiments, the fi rst cycle is a " blank" cycle (i.e., no fl uorescent oligonucleotides are added).…”

“…Since the CODEX method was first described in 2018 (17), this technology has been successfully adapted for use in FFPE tissues (16,18) and applied to immunophenotype solid (16) and hematological (19) malignancies. Establishment of a companion computational framework has been crucial for processing raw CODEX imaging data, mapping cellular interactions, and analyzing cellular neighborhoods (16)(17)(18)(19)(20)(21). These studies have accelerated the discovery of new immune cell subsets (16,17) and biomarkers (19), and correlated spatial organization with cancer prognosis (16) and immunotherapeutic outcomes (19).…”

Strategies for Accurate Cell Type Identification in CODEX Multiplexed Imaging Data

“…CODEX multiplexed imaging was done using a CODEX staining and imaging protocol previously described in detail (16,19). Settings used for the microscope are listed in Supplemental Table 1.…”

“…Incorrect celltype identification will lead to false interpretations of spatial features and study conclusions. Most studies using multiplexed imaging technologies have employed previously established pipelines created for non-imaging-based, single-cell-type identification, such as hand-gating flow plots or unsupervised clustering, and have used various methods of raw data processing and normalization (10,(15)(16)(17)(18)(19)(20).…”

Histological Studies of the Ventricular–Subventricular Zone as Neural Stem Cell and Glioma Stem Cell Niche

“…Sets of fluorescently labeled oligonucleotides complementary to sets of barcoded antibodies are administered, imaged, stripped, and repeated to measure up to 58 unique barcoded antibodies. 130,131 The resulting images are computationally registered and analyzed similarly to cyclic IF or IHC datasets, but have the advantage of having been stained together, removing variation in staining conditions, reagents, and time.…”